tbuckley/cabot-search huggingface dataset:
Abstract P2122: Coronary Heart Disease And Arthritis - An Analysis Of 2 Year Behavioral Risk Factor Surveillance System (BRFSS) Data
Authors: Akash Sharma, Velmurugan Anbu ananthan
Journal: Circulation Research (2023)

Introduction: Arthritis has increased systematic repercussions either by extra-articular manifestations or due to limited activities. Studies have fined an association between arthritis and heart disease. In this study, we try to find that arthritis is an independent association for myocardial infarction (MI) or coronary heart disease (CHD). Methods: In this study, we used the 2020 and 2021 Behavioral Risk Factor Surveillance System (BRFSS), a large, nationally representative, cross-sectional telephone survey. The data was divided into the participants having arthritis or no arthritis. Results were pooled and were represented with an odds ratio (OR) with a 95% CI interval. We used a logistic regression model to pool the results and adjusted for different variables. Two different models were constructed, and ANOVA was used to compare different models. The analysis was done by using R statistical software. Fisher t-test was used to compare between 2 groups. P-value &lt; 0.05 was considered significant. Results: Pooled results of the BRFSS survey led to 835,452 participants included in the final analysis. Of the included participant, 267020 has arthritis, and 568432 does not. 38.7% were males, and 61.3% were females. In the arthritis group, 38.7% were males, and 61.3% were females. Also, 55.1% of individuals in the arthritis group are older than 65. Our analysis found that arthritis people have a higher prevalence of diabetes and stroke. We analyzed logistic regression after adjusting for sex, race, age, diabetes, stroke, and BMI. It was found that participants with arthritis have higher odds of MI or CHD ( OR = 1.055, 95% CI 1.154-1.157, p-value &lt; 0.001) compared to non-arthritis people and when adjusted for sex, race, age it was 1.074, 95% CI 1.073-1.075, p-value&lt;0.001). Conclusion: We find that arthritis is associated with an increased risk of MI or CHD independent of other risk factors. Hence, patients with arthritis need close monitoring for heart-related disease.
Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes
Authors: Eric Schoger, Laura Stengel, Dominic Riedl, Lorenz H Lehmann, Rabea Hinkel, Samuel Sossalla, Laura C Zelarayan
Journal: Circulation Research (2023)

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.
Abstract P1027: Confocal And Mass Spectrometry-based Investigation Of REEP5 Depletion By AAV9 In The Mouse Heart
Authors: Michelle Di Paola, Cristine Reitz, Uros Kuzmanov, Kateleen Jia, Allen Teng, Anthony Gramolini
Journal: Circulation Research (2023)

The sarco(endo)plasmic reticulum (SR/ER) is an essential regulator of many key cellular processes, especially those that play a role in the development and progression of cardiac disease. However, many aspects of its structural organization remain poorly defined. Receptor Expression Enhancing Protein 5 (REEP5) is a cardiac enriched SR/ER membrane protein, which regulates organization of the highly differentiated SR/ER network and responses to stress. In zebrafish models, genetic knock-out of reep5 results in cardiac functional defects and reduced heart rate. Within the cardiomyocyte, depletion of Reep5 in vitro results in decreased muscle cell contraction, disrupted Ca 2+ signaling and SR/ER luminal vacuolization. For these studies, in vivo cardiac knock-down of Reep5 in the mouse was achieved using recombinant adeno-associated virus serotype 9 (rAAV9)-mediated gene delivery. Cardiac tissues or isolated cardiomyocytes were harvested at 7 days through to 4 weeks following knock-down, for biochemical and functional assessments. We observed that the largest significant change in REEP5 expression occurred 4 weeks post-rAAV9 injection, correlating to a 78% knock-down, observed by immunoblotting (unpaired t-test, p&lt;0.0001, n=6-8). 5 weeks following knock-down, mice developed lethal cardiac dysfunction. To assess the biochemical changes induced by Reep5 knock-down, we have established an organelle-specific cardiac proteomic profile of the microsome, mitochondria and cytosol, using subcellular fractionation and mass spectrometry (nLC-ESI-MS-HCD-MS). Coupled with high resolution confocal microscopy and 3D mapping techniques, we have examined localization and expression patterns of key SR/ER and mitochondrial proteins, that have altered expression following knock-down of Reep5 at the myocyte-level. These findings provide a detailed understanding of the role that REEP5 plays in maintaining ER homeostasis, SR/ER structure, and general organelle integrity. By identifying the mechanistic significance of REEP5 expression in the heart, we can work to delineate underappreciated pathways in cardiac muscle development.
Abstract P1103: A Combinatorial Stem Cell Therapy Using HiPSC-derived Cardiomyocytes And HiPSC-derived Mesenchymal Stem Cells For Cardiac Regeneration
Authors: Divya Sridharan, Syed Baseeruddin Alvi, Muhamad Mergaye, Abbey Forehand, Uzair Ahmed, Mahmood Khan
Journal: Circulation Research (2023)

Introduction: Myocardial infarction (MI) results in the death of nearly a billion cardiomyocytes in the heart. While conventional therapies alleviate the injury to some extent, they do not induce myocardial regeneration. On the other hand, transplantation of stem cells and their derived differentiated cells like hiPSC-derived cardiomyocytes (hiPSC-CMs) and hiPSC-derived mesenchymal stem cells (hiPSC-MSCs) have the potential to promote myocardial regeneration by direct (replacing dead cardiomyocytes) or indirect (paracrine) mechanisms, respectively. Hypothesis: We hypothesized that following co-transplantation of hiPSC-CMs and hiPSC-MSCs in ischemic hearts, hiPSC-MSCs will release paracrine factors to promote angiogenesis, reduce fibrosis, and improve cardioprotection and engraftment of transplanted hiPSC-CMs. Results: We cultured hiPSC-CMs and hiPSC-MSCs at different ratios, 1:1, 1:2 and 1:3, respectively. We observed improved contractility (MEA, and calcium transients) in the hiPSC-CM + hiPSC-MSC co-cultures, as compared to single cell-type cultures. Additionally, following exposure to hypoxia (1%O 2 ), we observed increased survival of hiPSC-CMs when co-cultured with hiPSC-MSCs (TUNEL assay). Furthermore, intramyocardial transplantation of (a) 1.5 х 10 6 hiPSC-CMs, (b) 1.5 х 10 6 hiPSC-MSCs or (c) 1.5 х 10 6 hiPSC-CMs + hiPSC-MSCs (3:1) in a mouse MI model, resulted in an improved cardiac function, LVEF (p &lt; 0.01) and FS (p&lt;0.01) in all animals receiving cell transplantation as compared to MI only group. Additionally, we observed efficient engraftment (immunostaining of human-specific markers, cTnT and HNA) of transplanted hiPSC-CMs in both hiPSC-CM-only and hiPSC-CM + hiPSC-MSC groups up to 4 weeks post-MI. We also observed increased neoangiogenesis (α-SMA, vWF) near the graft in the hiPSC-MSC-only and hiPSC-CM + hiPSC-MSC groups, as compared to control and hiPSC-CM-only groups. Lastly, a significant reduction in fibrosis (Masson-trichrome staining) was seen following transplantation of hiPSC-CMs + hiPSC-MSCs, compared to the control animals. Conclusion: Overall, our results established that a combinatorial stem therapy using hiPSC-CMs and hiPSC-MSCs for cardiac regeneration post-MI.
Abstract P2113: Zdhhc7 Uniquely Influences Cardiac Fibroblast Activity Amongst Golgi-localized S-acyltransferases
Authors: Robert Goldsmith, Rachel Scissors, Matthew Brody
Journal: Circulation Research (2023)

Cardiac fibroblasts (CF’s), the heart’s most critical regulators of the extra-cellular matrix, can undergo a continuum of activation states when stimulated that influence their proliferation, migration, collagen production, and cell-to-cell communication in response to injury. The signaling pathways that underlie this transformation though, are poorly understood. Previous studies have shown zDHHC3, an S-acyltransferase enzyme that mediates the reversible lipid modification, palmitoylation, to be important in cardiac remodeling in cardiomyocytes due to its ability to regulate intracellular signaling, though the role of zDHHC enzymes and palmitoylation in CF signaling are unknown. To investigate this, we deleted zDHHC3 or the closely related enzyme, zDHHC7, in cardiac fibroblasts to screen for changes in fibroblast activity and myocardial fibrosis. Loss of zDHHC7, but not zDHHC3, resulted in increased expression of the activated cardiac fibroblast marker α-smooth muscle actin in response to TGFb stimulation and a trend towards increased mRNA levels of fibrotic genes. More notably, zDHHC7 deletion significantly blunted migratory ability of adult cardiac fibroblasts in scratch assays. These data suggest zDHHC7 plays a critical role in CF signaling, and future experiments include analyzing the impact of zDHHC3/7 deletion alone or in combination on cardiac fibrosis and identifying key substrates and pathways being modified by these enzymes in CFs.
Abstract P1130: Enhanced Electrophysiological Maturation Of Human Pluripotent Stem Cell Derived Cardiomyocytes By An Optimal Combination Of Metabolic Modulators
Authors: Naoto Muraoka, Kris Alavattam, Xiulan Yang, Hans Reinecke, Jordan Klaiman, Charles Murry
Journal: Circulation Research (2023)

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) have emerged as ideal cell source for heart regeneration. Transplantation of hPSC-CMs to the damaged heart restores contractile function via electromechanical coupling between host and graft CMs. However, a few hurdles still remain in safety for clinical application, especially a transient period of ventricular arrhythmias after transplantation into large animals. Automaticity of graft cells, related to the electrical immaturity of hPSC-CMs, most likely triggers arrhythmic events. Here, we hypothesized that modulating metabolism toward an adult-like state would control ion channel gene expression and enhance electrophysiological maturation of hPSC-CMs in vitro. We systematically optimized combination of major metabolic components, including metabolic hormones, energy source and energy sensing, to achieve ion channel gene expression and electrophysiological phenotypes that more closely resemble adult cardiomyocytes. hPSC-CMs treated with optimized media displayed slower beat rates, longer field potentials, faster conduction velocities, and improved Ca 2+ kinetics. Moreover, optimized media increased mitochondrial content, respiratory capacity and fatty acid utilization for larger energy production, induced cell cycle exit, improved global transcriptome and sarcomere structure, and increased cell size. Subsequently, the matured cells were transplanted to mouse hearts, showing the engraftment comparable to immature hPSC-CMs. These results indicate that metabolic reprogramming can enhance many elements of maturation, including electrophysiology, myofibril organization, cell cycle, and capacity for oxidative phosphorylation.
Abstract P3203: Characterization Of Nrac, A Novel Cardiac And Adipose Enriched Microprotein
Authors: Taylor Coughlin, Cat A Makarewich
Journal: Circulation Research (2023)

Objective: Nrac ( n utritionally- r egulated a dipose and c ardiac-enriched gene) was previously identified as a microprotein encoded by the long noncoding RNA A530016L24Rik (human C14orf180) with 2 conserved transmembrane domains. Nrac is highly expressed in the heart as well as in brown and white adipose tissue and its expression is decreased in response to both fasting and obesity. To date, the biological function of Nrac is unknown, however a previous study indicated it may localize to the plasma membrane in white adipocytes. The goal of this study was to examine the molecular function of Nrac in adipocytes and the heart. Methods and Results: Using a brown adipocyte cell culture system, we differentiated pre-adipocytes to mature adipocytes and analyzed Nrac expression levels by qPCR. Nrac expression was undetectable in pre-adipocytes but markedly increased during differentiation and was robustly expressed in mature adipocytes. To determine the subcellular localization of Nrac, we transfected brown pre-adipocytes with a HA-V5-eptipe tagged Nrac fusion protein (HA-V5-Nrac), differentiated these cells to mature brown adipocytes, and performed immunostaining. These experiments showed that Nrac localizes specifically to the endoplasmic reticulum, and that overexpression of Nrac led to a striking increase in intracellular lipid droplet size as determined by BODIPY staining. To analyze Nrac in vivo, we generated a novel Nrac knockout (KO) mouse line using CRISPR/Cas9. Histological analysis of brown adipose tissue (BAT) from these mice revealed that intracellular lipid droplet size was markedly decreased in Nrac KO tissue compared to BAT from WT mice. RNA-sequencing analysis of heart tissue from WT and Nrac KO mice revealed a striking downregulation of genes related to adipogenesis and fatty acid metabolism in Nrac KO hearts and an upregulation in endoplasmic reticulum stress related genes. Conclusions: Our data indicate that Nrac is an endoplasmic reticulum membrane-localized microprotein that is highly expressed in the heart and adipose tissue. Our studies indicate that Nrac is involved in adipogenesis and lipid droplet formation. Future studies will aim to define the molecular mechanisms that contribute to Nrac function in heart and adipose tissue.
Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart
Authors: Anne Katrine Z Johansen, Ronald J Vagnozzi, Stephanie K Kidder Bowers, Kelly Grimes, Yasuhide Kuwabara, Michelle Sargent, Suh-chin Lin, Susan E Quaggin, Jeff D Molkentin
Journal: Circulation Research (2023)

Objective: Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Methods and Results: To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression in vivo . Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis. Conclusions: The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.
Abstract P1126: A Direct Comparison Of Cardiomyocyte Maturation In Vivo And In Vitro At Single Cell Resolution
Authors: Elaine Z Chen, Suraj Kannan, Renjun Zhu, Jason Guo, Chulan Kwon
Journal: Circulation Research (2023)

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer vast potential for clinical applications including cell therapy, drug screening, and disease modeling. However, in vitro -derived PSC-CMs incompletely mature with fetal-like phenotype, which drastically limits their clinical applications. Currently, it remains unknown why PSC-CMs fail to fully mature in culture. We sought to understand CM maturation process by generating high-quality single-cell RNA-sequencing (scRNA-seq) reference of in vivo mouse CMs sampled from embryonic stage to adult stage. Subsequently, we generated isogenic embryonic stem cells and established an in vitro scRNA-seq reference of differentiated CMs across a range of differentiation timepoints. By directly comparing the in vivo and the in vitro maturation trajectories, we identified a group of dysregulated transcription factors (TFs), NFE2L2, NRF1, MEF2A, JUN, YY1, ESRRA, SRF, SOX9, PPARA, which may be important to activate the CM maturation program. Therefore, we increased their levels in PSC-CMs and subsequently examined their transcriptome changes. Our initial analysis showed that higher levels of the TFs increase the expression of electron transport chain protein genes and decrease the expression of glycolysis protein genes, suggesting the initiation of metabolic maturation. Our study presents a first direct comparison of CM maturation in vivo and in vitro at single-cell resolution, which illuminates new research directions for understanding PSC-CM maturation.
Abstract P1107: Elmsan1 Regulates Differentiation And Maturation Of Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells
Authors: YU-AN LU, Jiacheng Sun, Lu Wang, Meimei Wang, Rachel Matthews, Jianyi J Zhang, Yang Zhou
Journal: Circulation Research (2023)

Introduction: ELMSAN1 is a scaffolding protein of the mitotic deacetylase complex (MiDAC) that is critical for early embryonic development. Studies on Elmsan1 knockout mice show that its absence results in heart malformation and embryo lethality. Despite this, the direct role of ELMSAN1 in heart development and formation remains unknown. To gain insights into this role, we differentiated human-induced pluripotent stem cell (hiPSC) into cardiomyocyte (hiPSC-CM) to mimic early cardiogenesis and assess the involvement of ELMSAN1 in CM maturation. Methods and Results: We utilized shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout techniques to generate ELMSAN1-deficient hiPSC, which were differentiated into CM for characterization. We showed that ELMSAN1 depletion caused a delay in the deactivation of pluripotency genes during early differentiation, resulting in decreased expression of cardiac-specific markers (TNNT2, MHY6), and reduced differentiation efficiency of CM. We also observed the impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels in ELMSAN1-deficient CM. Moreover, through a series of structural and functional assessments, we found these cells exhibited incomplete sarcomere Z-line structure and length (1.3 μm in KD vs. 1.5 μm in control), decreased calcium amplitude, increased time to peak value, faster beat rate with a shorter beat period, decreased field potential duration (300 ± 10 ms in KD vs. 500 ± 10 ms in control), and prolonged propagation delay. Importantly, we confirmed similar immaturity and functional changes in ELMSAN1 knockout CM, excluding the potential off-target effect. Mechanistically, we discovered that ELMSAN1 depletion resulted in decreased levels of H3K27ac in both hiPSC and CM, indicating an unexpected role of ELMSAN1 in maintaining global H3K27ac and gene expression. Conclusion: Our findings offer compelling evidence for the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC-CM. Notably, we were the first to identify the impact of ELMSAN1 on various aspects of hiPSC-CM generation, including cardiac differentiation, sarcomere formation, calcium handling, and electrophysiological maturation.